Question

Discuss in detail real time PCR (qPCR) including how PCR works, theory of qPCR, advantages, disadvantages, types of detection mechanisms used.

Let’s suppose that you were running RT-PCR and quatifying the level of a particular mRNA using qPCR. You run qPCR but you suspected that you are amplifying the wrong product.  What could you do to determine if the correct mRNA

Answer 1

QPCR and RT-PCR are both terms used in biotechnology and utilized for the production of multiple copies of DNA. ... RT-PCR is used to amplify the reversed transcription of the DNA code; QPCR measures the amplification. 3. RT-PCR is for amplification, while qPCR is for quantification.It required 1000 folds less template DNA or RNA for the reaction to occur as compared with the conventional PCR. Melting curve analysis: The main advantage of the quantitative PCR is the confirmation of the analytes through the melting curve analysis.Advantages. PCR has a number of advantages. It is fairly simple to understand and to use, and produces results rapidly. The technique is highly sensitive with the potential to produce millions to billions of copies of a specific product for sequencing, cloning, and analysis.

Real-Time Vs Traditional PCR Real-Time chemistries allow for the detection of PCR amplification during the early phases of the reaction. Measuring the kinetics of the reaction in the early phases of PCR provides a distinct advantage over traditional PCR detection. Traditional methods use Agarose gels for detection of PCR amplification at the final phase or end-point of the PCR reaction.

Limitations of End-Point PCR Agarose gel results are obtained from the end point of the reaction. Endpoint detection is very time consuming. Results may not be obtained for days. Results are based on size discrimination, which may not be very precise. As seen later in the section, the end point is variable from sample to sample. While gels may not be able to resolve these variabilities in yield, real-time PCR is sensitive enough to detect these changes. Agarose Gel resolution is very poor, about 10 fold. Real-Time PCR can detect as little as a two-fold change! Some of the problems with End-Point Detection: Poor Precision Low sensitivity Short dynamic range < 2 logs Low resolution Non - Automated Size-based discrimination only Results are not expressed as numbers Ethidium bromide for staining is not very quantitative Post PCR processing

There can be little doubt that the rapid and easy acquisition of quantitative mRNA expression data by real-time fluorescence-based RT-PCR has placed this technology at the centre of a revolution that is allowing researchers to probe cellular expression profiles with a very high degree of precision. However, the huge variability of samples and templates, the wide range of enzymes, primer, amplicon and chemistry combinations makes a comparison between results obtained in different laboratories difficult. The aim of this chapter was to delineate a series of steps that must be taken if the vast amount of quantitative data generated by qRT-PCR assays is to be not just precise but also biologically relevant.